Folate antagonists, particularly methotrexate, have become important drugs in the treatment of certain human malignancies. Methotrexate (4-amino-N.sup.10 -methylpteroylglutamic acid) is chemically similar to folic acid and its derivatives (particularly 5-methyltetrahydrofolate in human tissue) and interferes with folic acid and derivatives thereof to interrupt folate metabolism in the synthesis of DNA in the body. A specific example is the inhibition of enzyme dihydrofolate reductase caused by methotrexate. This process inhibits folate metabolism and thereby the synthesis of DNA.
The primary use of the enzyme is for the depletion of serum folates. A secondary use is in methotrexate rescue. Where methotrexate has worked in the body for an optimum time, it is necessary to stop its work in human tissue, since death is caused by too high a concentration of methotrexate in the human body for too long a period of time.
An enzyme, carboxypeptidase G.sub.1 (hereinafter designated as "G.sub.1 "), has been isolated which hydrolyzes the carboxyl-terminated glutamate from both reduced and nonreduced folate derivatives. The enzyme, G.sub.1, specifically breaks down the methotrexate and serum folates which then become inactive.
Attempts to purify the reaction mix and to obtain the particular carboxypeptidase G.sub.1 by conventional techniques have proved inadequate, probably due to the similar chemical structure and molecular weight of other protein materials in the crude reaction mixture. There is a need then to provide a simple, rapid and inexpensive method of separating carboxypeptidase G.sub.1 from the preparative reaction mix.
A method of preparing G.sub.1 containing a reaction mixture is described in the article "Purification and Properties of Carboxypeptidase G.sub.1," by J. L. McCullough et al, The Journal of Biological Science, Vol. 246, No. 23, pages 7207-7213, herein incorporated by reference.